A haplotype is the cis arrangement of two or more polymorphisms located on a single chromosome. Haplotype information is considered to be very useful because the haplotype is related to certain diseases or abnormalities, and specific drug sensitivities. Until now, haplotype analysis required tracking studies of genetic information about polymorphisms covering several generations of a family, and computing machine-based estimation algorithms. On the other hand, the advances in polymerization chain reaction (PCR) technology have enabled direct, molecular level analysis of DNA, and the use of the results of such analysis for determining the haplotype has been suggested. However, for determining the haplotype using PCR, it is necessary to use multiple combinations of primers specific to the allelic genes, and to carry out PCR a number of times. If we consider only 2 allelic genes, there have been attempts to determine the haplotype by positioning 2 primer pairs at the polymorphic sites and examining whether any amplification occurred. This method has been suggested for determination of the haplotype with a single test tube assay (see Japanese Patent Application Laid-Open No. 2002-272482). To be more specific, a forward primer containing the first polymorphism is modified with a label that yields different signals depending on the allele type on the 5′ end. Moreover, for the reverse primer containing the second polymorphism, a flap sequence that is not present in the target sequence is attached to the 5′ end, so that the primer is designed to yield an amplicon having a length that depends on the allele. Using these primers, allele-specific PCR assay is carried out from both the directions. Which primer pair caused amplification, can be known by detecting the fluorescent label in the PCR amplicon and from the length of the amplicon, and thus the haplotype can be determined.
In the above described known method, however, it is essential that the primers contain sites with the polymorphic sequences. This restricts the freedom of selecting the base sequence of the primers. Here, to carry out allele-specific PCR, the Tms of the primers used for the PCR have to be similar. However, with the above described restrictions in selecting the primer base sequences, it may be difficult in some cases to make the Tms of the primers similar. In other words, with the method of the Japanese Patent Application Laid-Open No. 2002-272482, highly accurate detection of the haplotype is likely to be restricted to cases where the polymorphisms are at locations for which primers of similar Tm can be used. Besides this, in this method of detection, it is difficult to shift the positions of the sequences of both the forward and reverse primers. Thus, if the forward and reverse primers form a primer dimer, it becomes impossible to detect the haplotype.